Investigating How Active Fluctuations Drive Immune Receptor Dynamics and Signaling

从细胞运动、细胞分裂和细胞生长到免疫反应的正常功能，力在驱动许多细胞功能方面都是重要的。细胞所产生的力在分子水平上发挥作用，改变关键信号分子的构象，启动细胞和组织尺度的信号转导途径，实现大规模的形态变形和集体运动。特别是，越来越清楚的是，细胞中的力不是静态的，而是由肌动蛋白、微管细胞骨架和分子马达的集体动力学驱动的强烈波动，这些分子马达在自然界中是高度非平衡的。大多数实验和理论工作都集中在证明生命系统中活跃涨落的存在和表征其物理含义。然而，关于活跃的波动如何在生理环境中调节细胞功能，人们知之甚少。在这个项目中，PI将确定在B细胞受体和T细胞受体介导的免疫反应信号的背景下，活跃的细胞骨架和力量波动的作用。PI的结果将在生物中力介导的分子转导过程中具有广泛的意义，如细胞黏附、迁移和发育过程。拟议工作的跨学科性质将加强我们对生命系统功能的基础物理学的理解，并在活性物质和细胞生物学领域产生重大影响。国际和平研究所将致力于将研究的关键方面与教学和外联活动结合起来。PI将提供批判性思维和研究实践的本科课程，使用有关主题的实践研究模块，如细胞力量生成。PI将把软物质和细胞生物学的概念引入教育专业的一门课程，向他们灌输对物理及其与生物的联系的欣赏，他们可以把这种欣赏带到小学课堂：这是一种潜在的强有力的方式，可以激励孩子们从事STEM职业。作为科学日计划的一部分，PI将在当地小学进行集中讨论细胞和软物质的物理方面的演讲。PI将积极招募不同的本科生群体，并参与吸引高中生从事生物物理研究的计划，作为已建立的暑期女孩计划和新提出的校园生物物理日的一部分。国际和平研究所还建议通过举办科学家以科学为灵感的照片和艺术家的绘画展览来加强推广科学与艺术的交叉点，向公众传播这些想法。这个项目是阐明非平衡波动对生命系统功能方面的生物学相关性的第一步。在这个项目中，PI将使用最先进的成像和计算分析来探索力波动在免疫反应中的作用，并使用两个例子。在目标1中，PI将检查B细胞中的受体聚集，这是抗原收集的重要第一步。目前的一个模型假设，肌动蛋白细胞骨架是BCR运动的扩散障碍。抗原结合和信号传递导致这些屏障的溶解和扩散受限的簇的生长。然而，PI的初步研究表明，肌动蛋白动力学可能在这一过程中发挥积极作用。活化膜-肌动蛋白相互作用的理论模型已被证明能够形成非平衡结构，支持受体大分子组装的形成。PI将使用单分子成像、分析和建模来测试BCR聚集性是否需要通过肌动蛋白动力学进行主动搅拌。在目标2中，PI将研究源自细胞内细胞骨架和运动动力学的力波动如何传递到细胞外环境并驱动信号转导。实验研究和理论工作表明，细胞骨架的非平衡涨落具有明显的非热特性，具有洛伦兹功率谱。另一方面，大多数关于TCR信号转导的研究都考虑了静态负荷下TCR抗原结合的动态变化。PI假设，由随机运动活动和肌动蛋白动力学引起的力波动有助于抗原识别和放大信号，并将使用牵引力显微镜、光遗传学和生物物理研究来绘制力波动的空间结构，并将其与信号动力学相关联，使她能够阐明这些非平衡波动对免疫细胞激活的作用。更广泛地说，这项工作将对理解生命系统如何活跃运作的基本原则产生影响。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Forces are important in driving many cellular functions from cell motion, cell division and cell growth to the proper functioning of the immune response. Cell generated forces have been implicated in playing a role both at the molecular scale for changing the conformation of key signaling molecules to initiate signal transduction pathways to the cellular and tissue scale for large scale morphological deformations and collective motion. In particular, it is becoming increasingly clear that forces in cells are not static, rather they show strong fluctuations driven by the collective dynamics of actin and microtubules cytoskeleton and molecular motors which are highly non-equilibrium in nature. Most experimental and theoretical work has focused on demonstrating the existence of active fluctuations in living systems and characterizing the physical implications. However, little is known about how active fluctuations regulate cell function in a physiological context. In this project the PI will determine the role of active cytoskeletal and force fluctuations in the context of the immune response for B cell receptor and T cell receptor mediated signaling. The PI's results will have broad implications in the field of force mediated molecular transduction processes in biology such as during cell adhesion, migration and development. The interdisciplinary nature of the proposed work will enhance our understanding of the fundamental physics underlying the function of living systems and have significant impact both in the fields of active matter and cell biology. The PI will work to integrate key aspects of the research with teaching and outreach activities. The PI will offer an undergraduate course on critical thinking and research practice using hands on research modules on relevant themes such as cellular force generation. The PI will introduce concepts from soft matter and cell biology into a course for education majors, to instill in them an appreciation for physics and its connection to biology, which they can take to elementary school classrooms: a potentially powerful way to inspire children to STEM careers. The PI will give talks focused on the physical aspects of cells and soft matter in local elementary schools as part of Science Day programs. The PI will actively recruit a diverse group of undergraduate students and participate in programs for attracting high school students to biological physics research as part of the established Summer Girls program and a newly proposed Biophysics Day on campus. The PI also proposes to enhance outreach by working on the intersection of Science and Art with an exhibition of science-inspired photographs by scientists and paintings by artists to disseminate these ideas to the general public.This project is a first step in elucidating the biological relevance of non-equilibrium fluctuations for functional aspects of living systems. In this project the PI will use state of the art imaging and computational analyses to explore the role of force fluctuations in the immune response using two examples. In Objective 1, the PI will examine receptor clustering in B cells, which is an essential first step in antigen gathering. A current model posits that the actin cytoskeleton acts as diffusive barriers to BCR movement. Antigen binding and signaling leads to dissolution of these barriers and diffusion-limited growth of the clusters. However, the PI's preliminary studies suggest that actin dynamics may have an active role in this process. Theoretical models of active membrane-actin interactions have been shown to be able to form non-equilibrium structures that can support the formation of macromolecular assemblies of receptors. The PI will use single molecule imaging, analysis and modeling to test whether BCR clustering requires active stirring by actin dynamics. In Objective 2, the PI will study how force fluctuations originating from cytoskeletal and motor dynamics inside cells are transmitted to the extracellular environment and drive signal transduction. Experimental studies and theoretical work have shown that the non-equilibrium cytoskeletal fluctuations are clearly athermal in character with a Lorentzian power spectrum. On the other hand, most studies of TCR signaling consider the dynamics of TCR-antigen bonds under static loads. The PI hypothesizes that force fluctuations arising from stochastic motor activity and actin dynamics facilitate antigen discrimination and amplify signaling and will use traction force microscopy, optogenetics and biophysical studies to map the spatial structure of force fluctuations and correlate it with the kinetics of signaling, enabling her to elucidate the role of these non- equilibrium fluctuations for immune cell activation. More generally the work will have implications in understanding the fundamental principles underlying how active living systems function.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.